Stimulation of cellular regeneration and differentiation in the inner ear

ABSTRACT

The present invention provides methods for stimulating the formation of inner ear cells, including inner ear sensory hair cells and inner ear support cells. The methods of the present invention damage and/or kill inner ear cells, and stimulate the formation of new, inner ear cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior application Ser. No.09/614,099, filed July 11, 2000, now abandoned, which is acontinuation-in-part of International Application No. PCT/US00/05736,filed Mar. 3, 2000, and International Application No. PCT/EP99/01153,filed Feb. 23, 1999.

FIELD OF THE INVENTION

The present invention relates to methods and compositions forstimulating the formation of inner ear cells, including inner earsensory hair cells and inner ear support cells.

BACKGROUND OF THE INVENTION

Sensorineuronal hearing loss (SNHL), also called “nerve deafness,” is asignificant communication problem that affects tens of millions ofpeople in the U.S. alone. Loss of the inner ear sensory hair cells thatdetect sound is thought to be a major cause of this deficit. The anatomyof the inner ear is well known to those of ordinary skill in the art(see, e.g., Gray's Anatomy, Revised American Edition (1977), pages859–867, incorporated herein by reference). In brief, the inner earincludes three sensory portions: the cochlea, which senses sound; thesemicircular canals, which sense angular acceleration; and the otolithicorgans, which sense linear acceleration. In each of these sensoryportions, specialized sensory hair cells are arrayed upon one or morelayers of inner ear supporting cells. Supporting cells underlie, atleast partially surround, and physically support sensory hair cellswithin the inner ear. In operation, the sensory hair cells arephysically deflected in response to sound or motion, and theirdeflection is transmitted to nerves which send nerve impulses to thebrain for processing and interpretation.

In mammals, the inner ear is normally incapable of regenerating damagedor dead inner ear sensory hair cells. Thus, hearing disorders thatresult from the death or deterioration of sensory hair cells typicallyresult in a permanent hearing impairment. Sensorineuronal hearing losscan be caused by a multitude of events including age-related loss(presbycusis), noise exposure, drug exposure (e.g., antibiotics andanti-cancer therapeutics), infections, genetic mutations (syndromic andnon-syndromic) and autoimmune disease.

Currently, the treatment for acquired sensorineuronal hearing lossinvolves the use of external hearing aids and cochlear implants. Bothdevices have rather limited therapeutic potential and more importantly,do not address the problem of restoring structure or function to theauditory sensory epithelium.

A more recent approach to the problem of regenerating sensory inner earhair cells is disclosed in published international application serialnumber PCT/US99/24829 which discloses methods for stimulating theregeneration of inner ear cells (including sensory hair cells) thatinclude the step of introducing into inner ear cells nucleic acidmolecules that encode a transcription factor capable of stimulating theregeneration of inner ear cells.

The present inventors have discovered that destruction of existing innerear sensory hair cells promotes the re-entry of normally quiescent innerear supporting cells (that express reduced levels of one or more cellcycle inhibitor proteins, or in which cell cycle protein activity hasbeen reduced) into the cell cycle to yield progeny cells that can beinduced to form inner ear sensory hair cells, as disclosed herein. Insome instances, destruction of existing inner ear sensory hair cells issufficient to stimulate underlying and/or surrounding inner ear supportcells to develop into sensory hair cells. In other instances, efficientregeneration of sensory hair cells from support cells requiresdestruction of existing inner ear sensory hair cells in combination withat least one other stimulus, as described herein. Additionally, thepresent inventors have discovered that stimulating the proliferation ofinner ear support cells (with or without stimulating the regeneration ofinner ear sensory hair cells) improves the auditory function of theinner ear.

SUMMARY OF THE INVENTION

The present invention provides methods for stimulating the formation ofinner ear cells, including inner ear sensory hair cells and inner earsupport cells. The methods of the present invention rely on theunexpected observation that damaging and/or killing inner ear cellsstimulates the formation of new, inner ear cells.

In one aspect, the present invention provides methods for stimulatingthe formation of inner ear sensory hair cells from inner ear supportcells. The methods of this aspect of the present invention include thestep (a) of damaging one or more inner ear sensory hair cells underconditions that promote the formation of one or more new sensory haircells from one or more support cells that are in contact with thedamaged sensory hair cell(s). Preferably a plurality of inner earsensory hair cells are formed from a plurality of inner ear supportcells. The methods of this aspect of the invention optionally includethe step (b) of further stimulating the formation of one or more innerear sensory hair cells from inner ear support cells that are in contactwith the damaged inner ear sensory hair cell. Step (b) can occur before,during, after or overlapping with step (a). In one embodiment, the stepof stimulating the formation of one or more inner ear sensory hair cellsfrom one or more inner ear support cells that are in contact with thedamaged inner ear sensory hair cell includes the steps of stimulatingthe inner car support cells to enter the cell cycle, then stimulating atleast some of the progeny of the inner ear support cells todifferentiate to form inner ear sensory hair cells.

Inner ear sensory hair cells can be damaged, for example, by contactwith an amount of an ototoxic agent, such as an antibiotic, preferablyan aminoglycoside antibiotic, that is effective to damage inner earsensory hair cells. The ototoxic agent can be introduced into the innerear by any art-recognized means, for example by injection (such as witha needle and syringe), or through a cannula. In one embodiment of thisaspect of the invention, inner ear sensory hair cells are sufficientlydamaged to cause their death.

In some embodiments of the present invention, damage inflicted on aninner ear sensory hair cell stimulates the formation of one or more newinner ear sensory hair cell from an inner ear support cell that is incontact with the damaged inner ear sensory hair cell. In otherembodiments, however, damage inflicted on an inner ear sensory hair cellis insufficient, by itself, to efficiently stimulate the formation ofone or more new inner ear sensory hair cells from an inner ear supportcell that is in contact with the damaged inner ear sensory hair cell.Thus, in one embodiment of the methods of this aspect of the presentinvention, the formation of inner ear sensory hair cells from inner earsupport cells is stimulated by damaging inner ear sensory hair cells andexpressing within inner ear support cells (before, during and/or afterthe step of damaging sensory hair cells) a transcription factor capableof stimulating inner ear sensory hair cells to form from inner earsupport cells. For example, in one embodiment of the present invention,a nucleic acid molecule encoding a transcription factor capable ofstimulating inner ear sensory hair cells to form from inner ear supportcells is introduced into inner ear support cells under conditions thatenable expression of the transcription factor. Representative examplesof transcription factors capable of stimulating the formation of innerear sensory hair cells from inner ear support cells include POU4F1,POU4F2, POU4F3, Brn3a, Brn3b and Brn3c.

In another embodiment of the methods of this aspect of the presentinvention, the formation of inner ear sensory hair cells from inner earsupport cells is stimulated by damaging inner ear sensory hair cells andinhibiting (before, during and/or after the step of damaging the sensoryhair cells) the expression of one or more cell cycle inhibitors activein inner ear support cells. Inhibitors of cell cycle inhibitors can besubstances, such as proteins, that act on the cell cycle inhibitordirectly or indirectly within the cell. By way of representativeexample, cell cycle inhibitors active in inner ear support cells includecyclin-dependent kinase inhibitors, such as cyclin-dependent kinaseinhibitors of the so-called CIP/KIP family including p21^(Cip1),p27^(Kip1) and p57^(Kip2). For example, the expression of a cell cycleinhibitor active in inner ear support cells can be inhibited byintroducing into inner ear support cells an expression vector thatexpresses a nucleic acid molecule that hybridizes under stringentconditions (such as stringency greater than 2×SSC at 55° C.) to anucleic acid molecule (such as an mRNA molecule) encoding a cell cycleinhibitor active in inner ear support cells.

In addition, various recombinant growth factors such as TGF-alpha,insulin and IGF-1 can be used to stimulate the formation of inner earsensory hair cells from inner ear support cells. A representative,effective concentration range for recombinant growth factors utilized invitro in the practice of the present invention is 1–1000ng/ml. Morespecifically, TGF-alpha is preferably used at an effective concentrationof from 1–100 ng/ml; insulin is preferably used at an effectiveconcentration of from 100–1000 ng/ml; and IGF-1 is preferably used at aneffective concentration of from 10–1000 ng/ml. For in vivo applications,a sufficient amount of recombinant growth factor would be administeredto produce the foregoing concentrations in vivo.

In preferred embodiments of this aspect of the invention, the formationof inner ear sensory hair cells from inner ear support cells results inimprovement in the auditory function of the treated inner ear. Thus, inone aspect, the invention provides methods for improving auditoryfunction in an inner ear comprising the steps of: (a) damaging a firstinner ear sensory hair cell under conditions that promote the formationof one or more new inner ear sensory hair cells from a support cell thatis in contact with the damaged, first inner ear sensory hair cell; and(b) measuring an improvement in auditory function in the inner eartreated in accordance with step (a).

In another aspect, the present invention provides methods forstimulating the formation of inner ear support cells. The methods ofthis aspect of the invention include the steps of damaging inner earsupport cells under conditions that promote the formation of new innerear support cells (for example by cell division of inner ear supportcells that are in contact with damaged inner ear support cells). In thisaspect of the invention, the inner ear support cell is damaged, and theformation of new inner ear support cells is stimulated, using the sametechniques described herein for the methods of the present inventionthat stimulate the formation of inner ear sensory hair cells from innerear support cells. Thus, for example, inner ear support cells can bedamaged by contact with an amount of an ototoxic agent, such as anaminoglycoside antibiotic, that is effective to damage inner ear supportcells. Again by way of example, new inner ear support cell formation canbe further stimulated by damaging inner ear support cells and expressing(before, during and/or after damaging inner ear support cells) withininner ear support cells a transcription factor (such as POU4F1, POU4F2,POU4F3, Brn3a, Brn3b and Brn3c) capable of stimulating inner ear supportcells to divide and form new inner ear support cells. In preferredembodiments of this aspect of the invention, the proliferation of innerear support cells results in improvement in the auditory function of thetreated inner ear.

The methods of the present invention are useful for stimulating theformation of inner ear cells, such as sensory hair cells and supportcells. Further, the methods of the present invention are useful toameliorate the symptoms of a hearing disorder in a mammal, such as ahuman, that is caused by the death or damage of inner ear cells.Additionally, the methods of the present invention can be used toidentify genes and/or proteins that are capable of stimulating theformation of inner ear support cells and/or the formation of inner earsensory hair cells from inner ear support cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows a cross section of the Organ of Corti.

FIG. 2 shows the number of BrdU-labeled, guinea pig JH4 cells followingserum deprivation for 24 hours and a BrdU pulse for the last 4 hours ofthe 24 hour period. Cells were counted under fluorescence microscopy.The combination of lipids and p27^(kip1) AS reversed growth arrest to40% of that seen with 10% FBS stimulation (p<0.0001). (+) FBS; (−) noFBS; (AS) antisense oligonucleotide; (lipid) lipofection.

FIG. 3 shows the ABR threshold of the right ears of mice two weeks afterthe inner ears of the mice had been treated with amikacin sulfate.Abbreviations are: ABR, auditory brainstem response; dB, decibels; SPL,sound pressure level; Wt, wild type; Het, p27 heterozygote; Ko, p27knock-out; kHZ, kilo Hertz.

FIG. 4 shows the ABR threshold of the left ears of mice two weeks afterthe inner ears of the mice had been treated with amikacin sulfate.Abbreviations are the same as those set forth in the description of FIG.3.

FIG. 5 shows the ABR threshold of the right ears of mice four weeksafter the inner ears of the mice had been treated with amikacin sulfate.Abbreviations are the same as for FIG. 3.

FIG. 6 shows the ABR threshold of the left ears of mice four weeks afterthe inner ears of the mice had been treated with amikacin sulfate.Abbreviations are the same as for FIG. 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein, the abbreviation “SSC” refers to a buffer used innucleic acid hybridization solutions. One liter of the 20× (twenty timesconcentrate) stock SSC buffer solution (pH 7.0) contains 175.3 g sodiumchloride and 88.2 g sodium citrate.

As used herein, the phrase “damaging one or more inner ear sensory haircells”, or “damaging a first inner ear sensory hair cell”, orgrammatical equivalents thereof, means causing a deleterious change inthe structure, biochemistry and/or physiology of the damaged, sensoryhair cell (including killing the damaged cell) compared to an inner earsensory hair cell that is cultured under substantially the sameconditions as the damaged cell, but which is not damaged.

As used herein, the phrase “improving auditory function” or “improvementin auditory function”, or grammatical equivalents thereof, meansimproving, by at least 10%, the sensitivity to sound of an inner ear bytreating the inner ear in accordance with the methods of the presentinvention, or effecting any measurable improvement in the sensitivity tosound of an inner ear that is completely unresponsive to sound prior totreatment in accordance with the present invention. The sensitivity tosound of the treated inner ear is measured by any art-recognized means(such as the auditory brainstem response) and compared to thesensitivity to sound of a control inner ear that is not treated inaccordance with the present invention and which is cultured undersubstantially the same conditions as the treated inner ear.

As applied to nucleic acid sequence comparisons or amino acid sequencecomparisons herein, the term “sequence homology” (also referred to as“sequence identity”) is defined as the percentage of amino acid residuesor nucleic acid residues in a subject amino acid sequence or nucleicacid sequence that are identical with part or all of a candidate aminoacid sequence or nucleic acid sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology(identity), and not considering any conservative substitutions as partof the sequence homology. Neither N- or C-terminal extensions norinsertions shall be construed as reducing homology. No weight is givento the number or length of gaps introduced, if necessary, to achieve themaximum percent homology (identity).

In one aspect, the present invention provides methods for stimulatingthe formation of inner ear sensory hair cells from inner ear supportcells. The methods of this aspect of the present invention include thestep (a) of damaging one or more inner ear sensory hair cells underconditions that promote the formation of one or more new sensory haircells from one or more support cells that are in contact with thedamaged sensory hair cell(s). Preferably a plurality of inner earsensory hair cells are formed from a plurality of inner ear supportcells. The methods of this aspect of the invention optionally includethe step (b) of further stimulating the formation of one or more innerear sensory hair cells from inner ear support cells that are in contactwith the damaged inner ear sensory hair cell. Step (b) can occur before,during, after or overlapping with step (a). The methods of this aspectof the present invention can be utilized in vivo and in vitro.

The anatomy of the inner ear is well known to those of ordinary skill inthe art (see, e.g., Gray's Anatomy, Revised American Edition (1977),pages 859–867, incorporated herein by reference). In particular, thecochlea includes the Organ of Corti which is primarily responsible forsensing sound. As shown in FIG. 1, the Organ of Corti 10 includes abasilar membrane 12 upon which are located a variety of supporting cells14, including border cells 16, inner pillar cells 18, outer pillar cells20, inner phalangeal cells 22, Dieter's cells 24 and Hensen's cells 26.Supporting cells 14 support inner hair cells 28 and outer hair cells 30.Tectorial membrane 32 is disposed above inner hair cells 28 and outerhair cells 30. The present invention is adapted, in one aspect, tostimulate regeneration of sensory hair cells 28 and 30 from underlyingsupporting cells 14. In another aspect, the present invention is adaptedto stimulate the formation of supporting cells 14.

The present inventors have observed that destruction of existing innerear sensory hair cells promotes the re-entry of normally quiescent innerear supporting cells into the cell cycle to yield progeny cells that canbe induced to form inner ear sensory hair cells as disclosed herein. Insome instances, destruction of existing inner ear sensory hair cells issufficient to stimulate underlying and/or surrounding inner ear supportcells to develop into sensory hair cells. In other instances, efficientregeneration of sensory hair cells from support cells requiresdestruction of existing inner ear sensory hair cells in combination withanother stimulus, as described herein.

In the practice of one aspect of the present invention, inner earsensory hair cells are damaged, for example by contact with an amount ofan ototoxic agent that is effective to damage inner ear sensory haircells. Representative examples of ototoxic agents useful for damaginginner ear sensory hair cells include aminoglycoside antibiotics (suchas, neomycin, gentamycin, streptomycin, kanamycin, amikacin andtobramycin). In the practice of the present invention, the foregoingaminoglycoside antibiotics are typically used in vitro at an effectiveconcentration in the range of from about 0.01 mM–10 mM, and in vivo atan effective concentration in the range of from about 100 to about 1,000milligrams per kilogram body weight per day (mg/kg/d). Additional,representative examples of chemical agents useful for damaging inner earsensory hair cells include the following anti-cancer agents: cisplatin,carboplatin and methotrexate which are typically used in vitro at aneffective concentration in the range of from about 0.01–0.1 mM, and invivo at an effective concentration in the range of from about 5 to about10 mg/kg/d. Other useful chemical agents include poly-L-lysine at aneffective concentration in the range of from about 0.1–1.0 mM in vitro,and magnesium chloride at an effective concentration in vitro in therange of from about 5–100 mM.

The ototoxic agent, or agents, can be introduced into the inner ear byany art-recognized means, for example by injection using a needle andsyringe, or by cochleostomy. Cochleostomy involves puncturing thecochlea and inserting a catheter through which a chemical agent can beintroduced into the cochlea. A cochleostomy method is disclosed, forexample, in Lalwani, A. K. et al., Hearing Research 114:139–147 (1997),which publication is incorporated herein by reference.

In one embodiment of the methods of the present invention, the formationof inner ear sensory hair cells from inner ear support cells isstimulated by damaging inner ear sensory hair cells and expressing(before, during, and/or after damaging the inner ear sensory hair cells)within at least some of the inner ear support cells a transcriptionfactor capable of stimulating the formation of an inner ear sensory haircell from an inner ear support cell. For example, in one embodiment, anucleic acid molecule encoding a transcription factor capable ofstimulating the formation of an inner ear sensory hair cell isintroduced into inner ear support cells under conditions that enableexpression of the transcription factor.

Transcription factors useful in this aspect of the present inventionhave the ability to stimulate regeneration of inner ear sensory haircells from inner ear supporting cells when utilized in the practice ofthe methods of the present invention. Some transcription factors usefulin this aspect of the present invention are required for the normaldevelopment, and/or for the normal functioning, of inner ear sensoryhair cells.

Representative examples of transcription factors useful in this aspectof the present invention include POU4F1 (Collum, R. G. et al., NucleicAcids Research 20(18):4919–4925 (1992)), POU4F2 (Xiang et al., Neuron11:689–701 (1993)), POU4F3 (Vahava, O., Science 279(5358):1950–1954(1998), Brn3a (also known as Brn3.0), Brn3b (also known as Brn3.2) andBrn3c (also known as Brn3.1) as disclosed in Gerrero et al., Proc.Nat'l. Acad. Sci. (U.S.A.) 90(22):10841–10845 (1993), Xiang, M. et al.,Proc. Nat'l. Acad. Sci. (U.S.A.) 93(21):11950–11955 (1996), Xiang, M. etal., J. Neurosci. 15(7Part 1):4762–4785 (1995), Erkman, L. et al.,Nature 381(6583):603–606 (1996), Xiang, M. et al., Proc. Nat'l. Acad.Sci. (U.S.A.) 94(17): 9445–9450 (1997), each of which publications isincorporated herein by reference. Some transcription factors useful inthis aspect of the present invention possess at least one homeodomainand/or at least one POU-specific domain, and have a molecular weight inthe range of from about 33 kDa to about 37 kDa.

As used herein, the term “homeodomain” means an amino acid sequence thatis at least 50% homologous (such as at least 75% homologous, or at least90% homologous) to the homeodomain amino acid sequence set forth in SEQID NO:1.

As used herein, the term “POU-specific domain” means an amino acidsequence that is at least 50% homologous (such as at least 75%homologous, or at least 90% homologous) to the POU-specific domain aminoacid sequence set forth in SEQ ID NO:2.

An example of an algorithm that can be used to determine the percentagehomology between two protein sequences, or between two nucleic acidsequences, is the algorithm of Karlin and Altschul (Proc. Natl. Acad.Sci. USA 87:2264–2268 (1990)), modified as in Karlin and Altschul (Proc.Natl. Acad. Sci. USA 90:5873–5877 (1993)). Such an algorithm isincorporated into the NBLEST and XBLEST programs of Altschul et al. (J.Mol. Biol. 215:403–410 (1990)).

Presently more preferred inner ear cell transcription factors useful inthe practice of the present invention are POU4F3 transcription factorhomologues (hereinafter referred to as POU4F3 homologues). POU4F3homologues useful in the practice of the present invention are capableof stimulating the regeneration of inner ear sensory hair cells fromsupporting cells and are at least 25% homologous (such as at least 50%homologous or at least 75% homologous, or at least 90% homologous) tothe POU4F3 transcription factor having the amino acid sequence set forthin SEQ ID NO:4 and which is encoded by the nucleic acid molecule of SEQID NO:3. As used herein, the term “POU4F3 homologues” includes thePOU4F3 protein having the amino acid sequence set forth in SEQ ID NO:4,which is the presently most preferred inner ear cell transcriptionfactor useful in the practice of the present invention. Representativeexamples of other POU4F3 homologues useful in the practice of thepresent invention are set forth in Xiang, M. et al., J. Neuroscience15(7):4762–4785 (1995), which publication is incorporated herein byreference.

Additional nucleic acid molecules encoding transcription factors usefulin the practice of the present invention can be isolated by using avariety of cloning techniques known to those of ordinary skill in theart. For example, cloned POU4F3 homologues cDNAs or genes, or fragmentsthereof, can be used as hybridization probes utilizing, for example, thetechnique of hybridizing radiolabeled nucleic acid probes to nucleicacids immobilized on nitrocellulose filters or nylon membranes as setforth at pages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual(2nd edition), J. Sambrook, E. F. Fritsch and T. Maniatis eds., thecited pages of which are incorporated herein by reference. Presentlypreferred hybridization probes for identifying additional nucleic acidmolecules encoding POU4F3 homologues are fragments, of at least 15nucleotides in length, of the cDNA molecule (or its complementarysequence) having the nucleic acid sequence set forth in SEQ ID NO:3,although the complete cDNA molecule having the nucleic acid sequence setforth in SEQ ID NO:3 is also useful as a hybridization probe foridentifying additional nucleic acid molecules encoding POU4F3 homologue.A presently most preferred hybridization probe for identifyingadditional nucleic acid molecules encoding POU4F3 homologues is theoligonucleotide having the nucleic acid sequence 5′-TAG AAG TGC AGG GCACGC TGC TCA TGG TAT G-3′ (SEQ ID NO:5).

Exemplary high stringency hybridization and wash conditions useful foridentifying (by Southern blotting) additional nucleic acid moleculesencoding POU4F3 homologues are: hybridization at 68° C. in 0.25 MNa₂HPO₄ buffer (pH 7.2) containing 1 mM Na₂EDTA, 20% sodium dodecylsulfate; washing (three washes of twenty minutes each at 65° C.) isconducted in 20 mM Na₂HPO₄ buffer (pH 7.2) containing 1 mM Na₂EDTA, 1%(w/v) sodium dodecyl sulfate.

Exemplary moderate stringency hybridization and wash conditions usefulfor identifying (by Southern blotting) additional nucleic acid moleculesencoding POU4F3 homologues are: hybridization at 45° C. in 0.25 MNa₂HPO₄ buffer (pH 7.2) containing 1 mM Na₂EDTA, 20% sodium dodecylsulfate; washing is conducted in 5×SSC, containing 0.1% (w/v) sodiumdodecyl sulfate, at 55° C. to 65° C.

Again, by way of example, nucleic acid molecules encoding transcriptionfactors useful in the present invention can be isolated by thepolymerase chain reaction (PCR) described in The Polymerase ChainReaction (K. B. Mullis, F. Ferre, R. A. Gibbs, eds), Birkhauser Boston(1994), incorporated herein by reference. Thus, for example, firststrand DNA synthesis can be primed using an oligo (dT) primer, andsecond strand cDNA synthesis can be primed using an oligonucleotideprimer that corresponds to a portion of the 5′-untranslated region of acDNA molecule that is homologous to the target DNA molecule. Subsequentrounds of PCR can be primed using the second strand cDNA synthesisprimer and a primer that corresponds to a portion of the 3′-untranslatedregion of a cDNA molecule that is homologous to the target DNA molecule.

By way of non-limiting example, representative PCR reaction conditionsfor amplifying nucleic acid molecules encoding transcription factorsuseful in the present invention are as follows. The following reagentsare mixed in a tube (on ice) to form the PCR reaction mixture: DNAtemplate (e.g., up to 1 μg genomic DNA, or up to 0.1 μg cDNA), 0.1–0.3mM dNTPs, 10 μl 10×PCR buffer (10×PCR buffer contains 500 mM KCl, 15 mMMgCl₂, 100 mM Tris-HCl, pH 8.3), 50 pmol of each PCR primer (PCR primersshould preferably be greater than 20 bp in length and have a degeneracyof 10² to 10³ ), 2.5 units of Taq DNA polymerase (Perkin Elmer, Norwalk,Conn.) and deionized water to a final volume of 50 μl. The tubecontaining the reaction mixture is placed in a thermocycler and athermocycler program is run as follows. Denaturation at 94° C. for 2minutes, then 30 cycles of: 94° C. for 30 seconds, 47° C. to 55° C. for30 seconds, and 72° C. for 30 seconds to two and a half minutes.

Preferably, PCR primers will be designed against conserved amino acidsequence motifs found in some or all of the known target proteinsequences. Examples of conserved amino acid sequence motifs againstwhich PCR primers can be designed for cloning additional POU4F3homologues are the POU-specific domain having the amino acid sequenceset forth in SEQ ID NO:2, and the homeodomain having the amino acidsequence set forth in SEQ ID NO:1.

Further, additional nucleic acid molecules encoding transcriptionfactors useful in the practice of the present invention can also beisolated, for example, by utilizing antibodies that recognizetranscription factor proteins. Methods for preparing monoclonal andpolyclonal antibodies are well known to those of ordinary skill in theart and are set forth, for example, in chapters five and six ofAntibodies A Laboratory Manual, E. Harlow and D. Lane, Cold SpringHarbor Laboratory (1988), the cited chapters of which are incorporatedherein by reference. By way of non-limiting example, antibodies weresuccessfully raised against a fusion protein constructed from theC-terminal end of Brn3 as described in Xiang, M. et al., J. Neuroscience15(7):4762–4785 (1995) and Xiang, M. et al., P.N.A.S. (U.S.A.)94:9445–9450 (1997), which publication is incorporated herein byreference.

Nucleic acid molecules that encode transcription factors useful in thepractice of the present invention can be isolated, for example, byscreening expression libraries. By way of non-limiting example, a cDNAexpression library can be screened using anti-POU4F3 homologueantibodies in order to identify one or more clones that encode a POU4F3homologue protein. DNA expression library technology is well known tothose of ordinary skill in the art. Screening cDNA expression librariesis fully discussed in Chapter 12 of Sambrook, J., Fritsch, E. F. andManiatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., the citedchapter of which is incorporated herein by reference.

By way of representative example, antigen useful for raising antibodiesfor screening expression libraries can be prepared in the followingmanner. A full-length cDNA molecule encoding a transcription factor,such as a POU4F3 homologue, (or a cDNA molecule that is not full-length,but which includes all of the coding region) can be cloned into aplasmid vector, such as a Bluescript plasmid (available from Stratagene,Inc., La Jolla, Calif.). The recombinant vector is then introduced intoan E. coli strain (such as E. coli XL1-Blue, also available fromStratagene, Inc.) and the protein encoded by the cDNA is expressed in E.coli and then purified. For example, E. coli XL 1-Blue harboring aBluescript vector including a cDNA molecule of interest can be grownovernight at 37° C. in LB medium containing 100 μg ampicillin/ml. A 50μl aliquot of the overnight culture can be used to inoculate 5 mg offresh LB medium containing ampicillin, and the culture grown at 37° C.with vigorous agitation to A₆₀₀=0.5 before induction with 1 mM IPTG.After an additional two hours of growth, the suspension is centrifuged(1000×g, 15 min, 4° C.), the media removed, and the pelleted cellsresuspended in 1 ml of cold buffer that preferably contains 1 mM EDTAand one or more proteinase inhibitors. The cells can be disrupted bysonication with a microprobe. The chilled sonicate is cleared bycentrifugation and the expressed, recombinant protein purified from thesupernatant by art-recognized protein purification techniques, such asthose disclosed in Methods in Enzymology, Vol. 182, Guide to ProteinPurification, Murray P. Deutscher, ed (1990), which publication isincorporated herein by reference.

Methods for preparing monoclonal and polyclonal antibodies are wellknown to those of ordinary skill in the art and are set forth, forexample, in chapters five and six of Antibodies A Laboratory Manual, E.Harlow and D. Lane, Cold Spring Harbor Laboratory (1988), the citedchapters of which are incorporated herein by reference. In onerepresentative example, polyclonal antibodies specific for a purifiedprotein can be raised in a New Zealand rabbit implanted with a whiffleball. One μg of protein is injected at intervals directly into thewhiffle ball granuloma. A representative injection regime is injections(each of 1 μg protein) at day 1, day 14 and day 35. Granuloma fluid iswithdrawn one week prior to the first injection (preimmune serum), andforty days after the final injection (postimmune serum).

Sequence variants, produced by deletions, substitutions, mutationsand/or insertions, of the transcription factors useful in the practiceof the present invention can also be used in the methods of the presentinvention. The amino acid sequence variants of the transcription factorsuseful in the practice of the present invention may be constructed bymutating the DNA sequences that encode the wild-type transcriptionfactor proteins, such as by using techniques commonly referred to assite-directed mutagenesis. Nucleic acid molecules encoding thetranscription factors useful in the practice of the present inventioncan be mutated by a variety of PCR techniques well known to one ofordinary skill in the art. (See, for example, the followingpublications, the cited portions of which are incorporated by referenceherein: “PCR Strategies”, M. A. Innis, D. H. Gelfand and J. J. Sninsky,eds., 1995, Academic Press, San Diego, Calif. (Chapter 14); “PCRProtocols: A Guide to Methods and Applications”, M. A. Innis, D. H.Gelfand, J. J. Sninsky and T. J. White, eds., Academic Press, N.Y.(1990)).

By way of non-limiting example, the two primer system utilized in theTransformer Site-Directed Mutagenesis kit from Clontech, may be employedfor introducing site-directed mutants into nucleic acid moleculesencoding transcription factors useful in the practice of the presentinvention. Following denaturation of the target plasmid in this system,two primers are simultaneously annealed to the plasmid; one of theseprimers contains the desired site-directed mutation, the other containsa mutation at another point in the plasmid resulting in elimination of arestriction site. Second strand synthesis is then carried out, tightlylinking these two mutations, and the resulting plasmids are transformedinto a mutS strain of E. coli. Plasmid DNA is isolated from thetransformed bacteria, restricted with the relevant restriction enzyme(thereby linearizing the unmutated plasmids), and then retransformedinto E. coli. This system allows for generation of mutations directly inan expression plasmid, without the necessity of subcloning or generationof single-stranded phagemids. The tight linkage of the two mutations andthe subsequent linearization of unmutated plasmids results in highmutation efficiency and allows minimal screening. Following synthesis ofthe initial restriction site primer, this method requires the use ofonly one new primer type per mutation site. Rather than prepare eachpositional mutant separately, a set of “designed degenerate”oligonucleotide primers can be synthesized in order to introduce all ofthe desired mutations at a given site simultaneously. Transformants canbe screened by sequencing the plasmid DNA through the mutagenized regionto identify and sort mutant clones. Each mutant DNA can then be fullysequenced or restricted and analyzed by electrophoresis on MutationDetection Enhancement gel (J. T. Baker) to confirm that no otheralterations in the sequence have occurred (by band shift comparison tothe unmutagenized control).

Again, by way of non-limiting example, the two primer system utilized inthe QuikChange™ Site-Directed Mutagenesis kit from Stratagene (La Jolla,Calif.), may be employed for introducing site-directed mutants intonucleic acid molecules encoding transcription factors useful in thepractice of the present invention. Double-stranded plasmid DNA,containing the insert bearing the target mutation site, is denatured andmixed with two oligonucleotides complementary to each of the strands ofthe plasmid DNA at the target mutation site. The annealedoligonucleotide primers are extended using Pfu DNA polymerase, therebygenerating a mutated plasmid containing staggered nicks. Aftertemperature cycling, the unmutated, parental DNA template is digestedwith restriction enzyme DpnI which cleaves methylated or hemimethylatedDNA, but which does not cleave unmethylated DNA. The parental, templateDNA is almost always methylated or hemimethylated since most strains ofE. coli, from which the template DNA is obtained, contain the requiredmethylase activity. The remaining, annealed vector DNA incorporating thedesired mutation(s) is transformed into E. coli.

In the design of a particular site directed mutagenesis experiment, itis generally desirable to first make a non-conservative substitution(e.g., Ala for Cys, His or Glu) and determine if activity is greatlyimpaired as a consequence. If the residue is by this means demonstratedto be important by activity impairment, or knockout, then conservativesubstitutions can be made, such as Asp for Glu to alter side chainlength, Ser for Cys, or Arg for His. For hydrophobic segments, it islargely size that is usefully altered, although aromatics can also besubstituted for alkyl side chains.

Other site directed mutagenesis techniques may also be employed withnucleic acid molecules encoding transcription factors useful in thepractice of the present invention. For example, restriction endonucleasedigestion of DNA followed by ligation may be used to generate deletionvariants of transcription factors useful in the practice of the presentinvention, as described in Section 15.3 of Sambrook et al. MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, New York, N.Y. (1989), incorporated herein by reference. Asimilar strategy may be used to construct insertion variants, asdescribed in section 15.3 of Sambrook et al., supra.

Oligonucleotide-directed mutagenesis may also be employed for preparingsubstitution variants of transcription factors useful in the practice ofthe present invention. It may also be used to conveniently prepare thedeletion and insertion variants of transcription factors useful in thepractice of the present invention. This technique is well known in theart as described by Adelman et al. (DNA 2:183 [1983]); Sambrook et al.,supra; “Current Protocols in Molecular Biology”, 1991, Wiley (NY), F. T.Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. D. Seidman, J. A.Smith and K. Struhl, eds., incorporated herein by reference.

Generally, oligonucleotides of at least 25 nucleotides in length areused to insert, delete or substitute two or more nucleotides in thenucleic acid molecules encoding transcription factors useful in thepractice of the present invention. An optimal oligonucleotide will have12 to 15 perfectly matched nucleotides on either side of the nucleotidescoding for the mutation. To mutagenize wild-type transcription factorproteins useful in the practice of the present invention, theoligonucleotide is annealed to the single-stranded DNA template moleculeunder suitable hybridization conditions. A DNA polymerizing enzyme,usually the Klenow fragment of E. coli. DNA polymerase I, is then added.This enzyme uses the oligonucleotide as a primer to complete thesynthesis of the mutation-bearing strand of DNA. Thus, a heteroduplexmolecule is formed such that one strand of DNA encodes the wild-typeprotein inserted in the vector, and the second strand of DNA encodes themutated form of the protein inserted into the same vector. Thisheteroduplex molecule is then transformed into a suitable host cell.

Mutants with more than one amino acid substituted may be generated inone of several ways. If the amino acids are located close together inthe polypeptide chain, they may be mutated simultaneously using oneoligonucleotide that codes for all of the desired amino acidsubstitutions. If, however, the amino acids are located some distancefrom each other (separated by more than ten amino acids, for example) itis more difficult to generate a single oligonucleotide that encodes allof the desired changes. Instead, one of two alternative methods may beemployed. In the first method, a separate oligonucleotide is generatedfor each amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions. An alternative methodinvolves two or more rounds of mutagenesis to produce the desiredmutant. The first round is as described for the single mutants:wild-type protein DNA is used for the template, an oligonucleotideencoding the first desired amino acid substitution(s) is annealed tothis template, and the heteroduplex DNA molecule is then generated. Thesecond round of mutagenesis utilizes the mutated DNA produced in thefirst round of mutagenesis as the template. Thus, this template alreadycontains one or more mutations. The oligonucleotide encoding theadditional desired amino acid substitution(s) is then annealed to thistemplate, and the resulting strand of DNA now encodes mutations fromboth the first and second rounds of mutagenesis. This resultant DNA canbe used as a template in a third round of mutagenesis, and so on.

Prokaryotes may be used as host cells for the initial cloning steps oftranscription factors useful in the practice of the present invention.They are particularly useful for rapid production of large amounts ofDNA, for production of single-stranded DNA templates used forsite-directed mutagenesis, for screening many mutants and/or putativeinner ear cell transcription factors simultaneously, and for DNAsequencing of the mutants generated. Suitable prokaryotic host cellsinclude E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110(ATCC No. 27,325) E. coli X1776 (ATCC No. 31,537), and E. coli B;however many other strains of E. coli, such as HB101, JM101, NM522,NM538, NM539, and many other species and genera of prokaryotes includingbacilli such as Bacillus subtilis, other enterobacteriaceae such asSalmonella typhimurium or Serratia marcesans, and various Pseudomonasspecies may all be used as hosts. Prokaryotic host cells or other hostcells with rigid cell walls are preferably transformed using the calciumchloride method as described in section 1.82 of Sambrook et al., supra.Alternatively, electroporation may be used for transformation of thesecells. Prokaryote transformation techniques are set forth in Dower, W.J., in Genetic Engineering, Principles and Methods, 12:275–296, PlenumPublishing Corp., 1990; Hanahan et al., Meth. EnzyMol. 204:63 (1991).

As will be apparent to those skilled in the art, any plasmid vectorscontaining replicon and control sequences that are derived from speciescompatible with the host cell may also be used to clone, express and/ormanipulate nucleic acid molecules encoding transcription factors usefulin the practice of the present invention. The vector usually has areplication site, marker genes that provide phenotypic selection intransformed cells, one or more promoters, and a polylinker regioncontaining several restriction sites for insertion of foreign DNA.Plasmids typically used for transformation of E. coli include pBR322,pUC18, pUC19, pUCI118, pUC119, and Bluescript M13, all of which aredescribed in sections 1.12–1.20 of Sambrook et al., supra. However, manyother suitable vectors are available as well. These vectors containgenes coding for ampicillin and/or tetracycline resistance which enablescells transformed with these vectors to grow in the presence of theseantibiotics.

The promoters most commonly used in prokaryotic vectors include theβ-lactamase (penicillinase) and lactose promoter systems (Chang et al.Nature 375:615 [1978]; Itakura et al., Science 198:1056 [1977]; Goeddelet al., Nature 281:544 [1979]) and a tryptophan (trp) promoter system(Goeddel et al., Nucl. Acids Res. 8:4057 [1980]; EPO Appl. Publ. No.36,776), and the alkaline phosphatase systems. While these are the mostcommonly used, other microbial promoters have been utilized, and detailsconcerning their nucleotide sequences have been published, enabling askilled worker to ligate them functionally into plasmid vectors (seeSiebenlist et al., Cell 20:269 [1980]).

The construction of suitable vectors containing DNA encoding replicationsequences, regulatory sequences, phenotypic selection genes and the DNAencoding a transcription factor useful in the practice of the presentinvention are prepared using standard recombinant DNA procedures.Isolated plasmids and DNA fragments are cleaved, tailored, and ligatedtogether in a specific order to generate the desired vectors, as is wellknown in the art (see, for example, Sambrook et al., supra).

In another embodiment of the methods of the present invention, theformation of inner ear sensory hair cells from inner ear support cellsis stimulated by damaging inner ear sensory hair cells and inhibitingthe expression (before, during and/or after damaging the inner earsensory hair cells) of one or more cell cycle inhibitors active in innerear support cells. In this way, inner ear support cells that are incontact with damaged sensory hair cells can be stimulated to divide andat least some of the resulting progeny form inner ear sensory haircells. By way of representative example, cell cycle inhibitors active ininner ear support cells include cyclin-dependent kinase inhibitors, suchas cyclin-dependent kinase inhibitors of the so-called CIP/KIP familyincluding p21^(Cip1), p27^(Kip1) and p57^(Kip2).

Specific examples of cell cycle inhibitors active within inner earsupport cells include: p57^(Kip2) (Lee et al., Genes Dev. 9(6):639–649(1995)(SEQ ID NO:6)); p27^(Kip1) (Cell 78(1):59–66 (1994)(SEQ ID NOS:8and 9)); p21^(Cip1) (El-Diery et al., Cell 75(4):817–825 (1993)(SEQ IDNOS:10 and 11)); p19 Ink 4d (Chan et al., Mol. Cell. Biol.15(5):2682–2688 (1995)(SEQ ID NOS:12 and 13)); p18 Ink 4c (Guan et al.,Genes Dev. 8(24):2939–2952 (1994)(SEQ ID NOS:14 and 15)); p15 Ink 4b(Hannon and Beach, 371(6494):257–261 (1994)(SEQ ID NOS:16 and 17)); andp16 Ink 4a (Serrano, M. et al., Nature 366(6456):704–707 (1993)(SEQ IDNOS:18 and 19)). Nucleic acid molecules that encode cell cycleinhibitors useful in the practice of the present invention hybridize tothe antisense strands of any one of the nucleic acid molecules set forthin SEQ ID NOS: 6, 8, 10, 12, 14, 16 and 18 under at least onehybridization stringency greater than 2×SSC at 55° C., such as 1×SSC at60° C., or 0.2×SSC at 60° C.

Inhibitors of cell cycle inhibitors can be substances, such as proteins,that act on the cell cycle inhibitor in an intracellular, direct orindirect manner. Additionally, inhibitors of cell cycle inhibitors canbe antisense nucleic acid molecules that are complementary to all or aportion of a nucleic acid molecule (such as an mRNA molecule) thatencodes a cell cycle inhibitor protein, and that hybridize to thenucleic acid molecule encoding a cell cycle inhibitor protein understringent conditions (such as a stringency greater than 2×SSC at 55° C.,e.g., 1×SSC at 60° C. or 0.2×SSC at 60° C.).

Any art-recognized method can be used to inhibit cell cycle inhibitorgene expression in inner ear support cells. For example, the expressionof a cell cycle inhibitor active in inner ear support cells can beinhibited by introducing into inner ear support cells a vector thatincludes a portion (or all) of a nucleic acid molecule, in antisenseorientation relative to a promoter sequence, that encodes a cell cycleinhibitor active in inner ear support cells.

In general, antisense technology utilizes a DNA sequence that isinverted relative to its normal orientation for transcription and soexpresses an RNA transcript that is complementary to a target mRNAmolecule expressed within the host cell (i.e., the RNA transcript of theanti-sense gene can hybridize to the target mRNA molecule throughWatson-Crick base pairing). An anti-sense gene may be constructed in anumber of different ways provided that it is capable of interfering withthe expression of a target gene, such as a gene encoding a cell cycleinhibitor. The anti-sense gene can be constructed by inverting thecoding region (or a portion thereof) of the target gene relative to itsnormal orientation for transcription to allow the transcription of itscomplement, hence the RNAs encoded by the anti-sense and sense gene arecomplementary.

The anti-sense gene generally will be substantially identical to atleast a portion of the target gene or genes. The sequence, however, neednot be perfectly identical to inhibit expression. Generally, higherhomology can be used to compensate for the use of a shorter anti-sensegene. The minimal identity will typically be greater than about 65%, buta higher identity might exert a more effective repression of expressionof the endogenous sequences. Substantially greater identity of more thanabout 80% is preferred, though about 95% to absolute identity would bemost preferred.

Furthermore, the anti-sense gene need not have the same intron or exonpattern as the target gene, and non-coding segments of the target genemay be equally effective in achieving anti-sense suppression of targetgene expression as coding segments. Normally, a DNA sequence of at leastabout 30 or 40 nucleotides should be used as the anti-sense gene,although a longer sequence is preferable. The construct is thenintroduced into one or more inner ear support cells and the antisensestrand of RNA is produced.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of target genes. It is possible to design ribozyme transgenesthat encode RNA ribozymes that specifically pair with a target RNA andcleave the phosphodiester backbone at a specific location, therebyfunctionally inactivating the target RNA. In carrying out this cleavage,the ribozyme is not itself altered, and is thus capable of recycling andcleaving other molecules. The inclusion of ribozyme sequences withinantisense RNAs confers RNA-cleaving activity upon them, therebyincreasing the activity of the antisense constructs. Tabler et al.(1991, Gene 108:175) have greatly simplified the construction ofcatalytic RNAs by combining the advantages of the anti-sense RNA and theribozyme technologies in a single construct. Smaller regions of homologyare required for ribozyme catalysis, therefore this can promote therepression of different members of a large gene family if the cleavagesites are conserved.

An additional strategy suitable for suppression of target gene activityentails the sense expression of a mutated or partially deleted form ofthe protein encoded by the target gene according to general criteria forthe production of dominant negative mutations (Herskowitz I, Nature329:219–222 (1987)).

Any art-recognized gene delivery method can be used to introduce anucleic acid molecule encoding a transcription factor (or a vectorincluding an antisense DNA molecule) into inner ear cells for expressiontherein, including: direct injection, electroporation, virus-mediatedgene delivery, amino acid-mediated gene delivery, biolistic genedelivery, lipofection and heat shock. Non-viral methods of gene deliveryinto inner ear cells are disclosed in Huang, L., Hung, M-C, and Wagner,E., Non-Viral Vectors for Gene Therapy, Academic Press, San Diego,Calif. (1999), which is incorporated herein by reference.

For example, genes can be introduced into cells in situ, or afterremoval of the cells from the body, by means of viral vectors. Forexample, retroviruses are RNA viruses that have the ability to inserttheir genes into host cell chromosomes after infection. Retroviralvectors have been developed that lack the genes encoding viral proteins,but retain the ability to infect cells and insert their genes into thechromosomes of the target cell (A. D. Miller, Hum. Gen. Ther. 1:5–14(1990)).

Adenoviral vectors are designed to be administered directly to patients.Unlike retroviral vectors, adenoviral vectors do not integrate into thechromosome of the host cell. Instead, genes introduced into cells usingadenoviral vectors are maintained in the nucleus as an extrachromosomalelement (episome) that persists for a limited time period. Adenoviralvectors will infect dividing and non-dividing cells in many differenttissues in vivo including airway epithelial cells, endothelial cells,hepatocytes and various tumors (B. C. Trapnell, Adv Drug Del Rev.12:185–199 (1993)).

Another viral vector is the herpes simplex virus, a large,double-stranded DNA virus that has been used in some initialapplications to deliver therapeutic genes to neurons and couldpotentially be used to deliver therapeutic genes to some forms of braincancer (D. S. Latchman, Mol. Biotechnol. 2:179–95 (1994)). Recombinantforms of the vaccinia virus can accommodate large inserts and aregenerated by homologous recombination. To date, this vector has beenused to deliver interleukins (ILs), such as human IL-1β and thecostimulatory molecules B7-1 and B7-2 (G. R. Peplinski et al., Ann.Surg. Oncol. 2:151–9 (1995); J. W. Hodge et al., Cancer Res. 54:5552–55(1994)).

Another approach to gene therapy involves the direct introduction of DNAplasmids into patients. (F. D. Ledley, Hum. Gene Ther. 6:1129–1144(1995)). The plasmid DNA is taken up by cells within the body and candirect expression of recombinant proteins. Typically plasmid DNA isdelivered to cells in the form of liposomes in which the DNA isassociated with one or more lipids, such as DOTMA(1,2,-diolcyloxypropyl-3-trimethyl ammonium bromide) and DOPE(dioleoylphosphatidylethanolamine). Formulations with DOTMA have beenshown to provide expression in pulmonary epithelial cells in animalmodels (K. L. Brigham et al., Am. J. Med. Sci. 298:278–281 (1989); A. B.Canonico et al., Am. J. Respir. Cell. Mol. Biol. 10:24–29 (1994)).Additionally, studies have demonstrated that intramuscular injection ofplasmid DNA formulated with 5% PVP (50,000 kDa) increases the level ofreporter gene expression in muscle as much as 200-fold over the levelsfound with injection of DNA in saline alone (R. J. Mumper et al., Pharm.Res. 13:701–709 (1996); R. J. Mumper et al., Proc. Intern. Symp. Cont.Rol. Bioac. Mater. 22:325–326 (1995)). Intramuscular administration ofplasmid DNA results in gene expression that lasts for many months (J. A.Wolff et al., Hum. Mol. Genet. 1:363–369 (1992); M. Manthorpe et al.,Hum. Gene Ther. 4:419–431 (1993); G. Ascadi et al., New Biol. 3:71–81(1991), D. Gal et al., Lab. Invest. 68:18–25 (1993)).

Additionally, uptake and expression of DNA has also been observed afterdirect injection of plasmid into the thyroid (M. Sikes et al., Hum. GeneTher. 5:837–844 (1994)) and synovium (J. Yovandich et al., Hum. GeneTher. 6:603–610 (1995)). Lower levels of gene expression have beenobserved after interstitial injection into liver (M. A. Hickman et al.,Hum. Gene Ther. 5:1477–1483 (1994)), skin (E. Raz et al., Proc. Natl.Acad. Sci. 91:9519–9523 (1994)), instillation into the airways (K. B.Meyer et al., Gene Therapy 2:450–460 (1995)), application to theendothelium (G. D. Chapman et al., Circulation Res. 71:27–33 (1992); R.Riessen et al., Human Gene Therapy 4:749–758 (1993)), and afterintravenous administration (R. M. Conry et al., Cancer Res. 54:1164–1168(1994)).

Various devices have been developed for enhancing the availability ofDNA to the target cell. A simple approach is to contact the target cellphysically with catheters or implantable materials containing DNA (G. D.Chapman et al., Circulation Res. 71:27–33 (1992)). Another approach isto utilize needle-free, jet injection devices which project a column ofliquid directly into the target tissue under high pressure. (P. A. Furthet al., Anal Biochem. 20:365–368 (1992); (H. L. Vahlsing et al., J.Immunol. Meth. 175:11–22 (1994); (F. D. Ledley et al., Cell Biochem.18A:226 (1994)).

Another device for gene delivery is the “gene gun” or Biolistic™, aballistic device that projects DNA-coated micro-particles directly intothe nucleus of cells in vivo. Once within the nucleus, the DNA dissolvesfrom the gold or tungsten microparticle and can be expressed by thetarget cell. This method has been used effectively to transfer genesdirectly into the skin, liver and muscle (N. S. Yang et al., Proc. Natl.Acad. Sci. 87:9568–9572 (1990); L. Cheng et al., Proc. Natl. Acad. Sci.USA. 90:4455–4459 (1993); R. S. Williams et al., Proc. Natl. Acad. Sci.88:2726–2730 (1991)).

Cochleostomy involves puncturing the cochlea and inserting a catheterthrough which a chemical agent, such as a nucleic acid molecule, can beintroduced into the cochlea. A cochleostomy method is disclosed, forexample, in Lalwani, A. K. et al., Hearing Research 114:139–147 (1997),which publication is incorporated herein by reference.

Another approach to targeted gene delivery is the use of molecularconjugates, which consist of protein or synthetic ligands to which anucleic acid- or DNA-binding agent has been attached for the specifictargeting of nucleic acids to cells (R. J. Cristiano et al., Proc. Natl.Acad. Sci. USA 90:11548–52 (1993); B. A. Bunnell et al., Somat. CallMol. Genet. 18:559–69 (1992); M. Cotten et al., Proc. Natl. Acad. Sci.USA 89:6094–98 (1992)). Once the DNA is coupled to the molecularconjugate, a protein-DNA complex results. This gene delivery system hasbeen shown to be capable of targeted delivery to many cell types throughthe use of different ligands (R. J. Cristiano et al., Proc. Natl. Acad.Sci. USA 90:11548–52 (1993)). For example, the vitamin folate has beenused as a ligand to promote delivery of plasmid DNA into cells thatoverexpress the folate receptor (e.g., ovarian carcinoma cells) (S.Gottschalk et al., Gene Ther. 1:185–91 (1994)). The malariacircumsporozoite protein has been used for the liver-specific deliveryof genes under conditions in which ASOR receptor expression onhepatocytes is low, such as in cirrhosis, diabetes, and hepatocellularcarcinoma (Z. Ding et al., J. Biol. Chem. 270:3667–76 (1995)). Theoverexpression of receptors for epidermal growth factor (EGF) on cancercells has allowed for specific uptake of EGF/DNA complexes by lungcancer cells (R. Cristiano et al., Cancer Gene Ther. 3:4–10 (1996)). Thepresently preferred gene delivery method is lipofection.

When the methods of the present invention are utilized in vitro, thewhole inner ear, including the Organ of Corti, is preferably excised andcultured and manipulated in a culture vessel. Presently preferredembodiments of an apparatus that is useful for culturing inner ears invitro are disclosed in U.S. patent No. 5,437,998; U.S. Pat. No.5,702,941 and U.S. Pat. Ser. No. 5,763,279, each of which isincorporated herein by reference.

In general, presently preferred embodiments of an apparatus forculturing inner ears include a gas permeable bioreactor comprising atubular vessel with walls that may be constructed at least partially ofa gas permeable material, such as silicone rubber. The vessel in onepreferred embodiment is constructed such that half of it is comprised ofgas permeable material and the remaining portion is made of nonpermeablematerial. The gas permeable materials commonly available are opaque.Thus, using nonpermeable material for at least part of the bioreactormay provide an advantage in allowing visual inspection of the tubularvessel chamber.

The tubular vessel has closed ends, a substantially horizontallongitudinal central axis, and one or more vessel access ports. Thevessel access ports provide access to the bioreactor for input of mediumand cells, and for removal of old medium from the tubular vessel. Thisis easily done through the vessel access ports which are also referredto as valves or syringe ports. In the preferred embodiment, the vesselaccess ports are constructed of valves with syringe ports.

Preferably the vessel is rotatable about its horizontal longitudinalcentral axis. A preferred means for rotation is a motor assembly whichsits on a mounting base and has means for attachment to the tubularvessel. The speed of rotation can be adjusted so that the inner earwithin the tubular vessel is constantly in motion, but rotation of thetubular vessel should not be fast enough to cause significant turbulencein the aqueous medium within the tubular vessel.

If so desired, the use of gas permeable material in the construction ofat least part of the tubular vessel wall permits O₂ to diffuse throughthe vessel walls and into the cell culture media in the vessel chamber.Correspondingly, CO₂ diffuses through the walls and out of the vessel.Thus, the use of gas permeable material in the construction of at leastpart of the tubular vessel wall typically overcomes the need for airinjection into the bioreactor vessel. Air injection into the aqueousmedium within the bioreactor vessel may be utilized, however, ifadditional oxygen is required to culture an inner ear. When an air pumpis utilized to inject air into the aqueous medium, an air filter is alsoemployed to protect the air pump valves from dirt.

An alternative embodiment of the bioreactor useful in the practice ofthe present invention is an annular vessel with walls that may beconstructed at least partially of a gas permeable material. Annular isdefined herein to include annular, toroidal and other substantiallysymmetrical ring-like shaped tubular vessels. The annular vessel hasclosed ends and a substantially horizontal longitudinal central axis.

In another embodiment, the bioreactor useful in the practice of thepresent invention comprises a tubular vessel constructed at leastpartially of a gas permeable material. The vessel has closed ends and asubstantially horizontal longitudinal central axis around which itrotates. The vessel furthermore has two slidably interconnected memberswherein a first member fits slidably into a second member, forming aliquid tight seal therebetween and providing a variable volume tubularvessel. The bioreactor has means for rotating the tubular vessel aboutits substantially horizontal longitudinal central axis. One or morevessel access ports are provided for transferring materials into and outof the vessel.

In situations where minimization of contamination is necessary (e.g.,AIDS or human tissue research), disposability of the bioreactor usefulin the practice of the present invention is a particular advantage.Moreover, the embodiment of the bioreactor with slidably interconnectedmembers may be adjusted to provide the exact size bioreactor needed.

Presently preferred, commercially available bioreactors useful in thepractice of the present invention for culturing fluid-filled sensoryorgans are known as the High Aspect Ratio Vessel (HARV™) and theCylindrical Cell Culture Vessel (CCCV™) and are manufactured bySynthecon, Inc. (8054 El Rio, Houston, Tex.).

Neuralbasal™ media from Gibco BRL (Gibco BRL media are produced by LifeTechnologies, Corporate Headquarters, Gaithersburg, Md.), which requiresthe addition of B27 or N2 media supplement, is the presently preferredculture medium for culturing inner ears in vitro. Other culture mediacan be successfully used, however, to culture fluid-filled sensoryorgans in the practice of the present invention. Other suitable mediainclude DME, BME and M-199 with fetal calf serum or horse serum. All ofthe foregoing media are sold by Gibco -BRL. When using Neuralbasal™medium, N2 or B27 supplements play a more significant role when extendedperiods of culture (>96 hr) are attempted.

In another aspect, the present invention provides methods forstimulating the formation of inner ear support cells. The methods ofthis aspect of the invention include the steps of damaging inner earsupport cells under conditions that promote the formation of new innerear support cells (for example by cell division of inner ear supportcells that are in contact with damaged inner ear support cells). In thisaspect of the invention, the inner ear support cell is damaged, and theformation of new inner ear support cells is stimulated, using the sametechniques described herein for the methods of the present inventionthat stimulate the formation of inner ear sensory hair cells from innerear support cells. Thus, for example, inner ear support cells can bedamaged by contact with an amount of an ototoxic agent, such as anaminoglycoside antibiotic, that is effective to damage inner ear supportcells. Again by way of example, new inner ear support cell formation canbe further stimulated by damaging inner ear support cells and expressing(before, during and/or after damaging inner ear support cells) withininner ear support cells a transcription factor (such as POU4F1, POU4F2,POU4F3, Brn3a, Brn3b and Brn3c) capable of stimulating inner ear supportcells to divide and form new inner ear support cells. In preferredembodiments of this aspect of the invention, the proliferation of innerear support cells results in improvement in the auditory function of thetreated inner ear.

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention.

EXAMPLE 1 Overexpressing POU4F3 in Immortalized Supporting-Cell Lines InVitro.

POU4F3 is a DNA binding transcription factor exhibiting remarkablespecificity to the hair-cells in the inner ear. Mutations in POU4F3 areknown to cause developmental failures in mice, and hearing loss in bothmice and humans. The role of POU4F3 in directing the development ofhair-cell precursors is investigated by transfecting inner earsupporting-cell lines with POU4F3.

To detect the expression of POU4F3 in live cultures, the expression ofEnhanced Green Fluorescent Protein (EGFP), translated from a bicistronicmRNA which includes both POU4F3 and GFP coding regions, is monitored.Specifically, a 1250 bp cDNA encoding POU4F3 including 70 bp of 5′UTRand 73 bp of 3′UTR is directionally cloned into the unique EcoRVrestriction site of the pIRES2-EGFP vector (Clonetech) directly downstream from the human CMV major immediate early promoter/enhancer. Anintervening synthetic intron is cloned downstream from the POU4F3 geneto enhance the stability of the mRNA. The internal ribosomal entry site(IRES) from encephelomyocarditis virus is cloned between the POU4F3 geneand the GFP gene to allow for the translation of GFP and POU4F3 proteinfrom the same mRNA. Immediately following the GFP coding region is apoly-adenylation signal from the bovine growth hormone gene. Thisexpression cassette is designed to take advantage of the bicistronicpromoter to allow tracking of transfection and expression of POU4F3 andGFP by visualization of expressed GFP under fluorescence microscopy.This second generation GFP vector has a red shifted variant of wild-typeGFP (Excitation maximum=488 nm; emission maximum=507 nm) which has beenoptimized for brighter fluorescence. To optimize the identification ofcells expressing high levels of the POU4F3 protein, the pIRES2 vectorutilizes a partially disabled IRES sequence (Rees, S. et al.,BioTechniques 20:102110 (1996)). This attenuated IRES leads to a reducedrate of translation initiation at the GFP start codon relative to thatof the POU4F3 gene.

The supporting-cell line (UCL) was established from the inner earvestibular sensory epithelia of the 112k^(b)-tsA 58 transgenic mouse(Immortamouse). Utricles from postnatal day one mice were dissected andthe sensory epithelia (hair-cells and supporting-cells) isolated afterbrief thermolysin treatment at 37° C. The resultant supporting-cell linewas derived after several passages at permissive conditions (33° C. andγINF) which stimulates rapid cell proliferation. Confluence was achievedin 3–4 days. This process resulted in the death of all hair-cells asdetermined by ICC and electron microscopy (EM). The UCLs were furthercharacterized by an antibody called ZO-1 (which labels tight junctionsthat are present in supporting-cells) and EM (which showed tightjunction complexes, secretory vesicles and luminal surface microvilli,all characteristic of supporting-cells).

Culture medium for the UCL cell line consisted of DMEM/F12 (Gibco),fetal bovine serum (10%) and γINF (20 u/ml). Media changes were done 2to 3 times a week depending on the growth rate of the cells. Single-cellclones were developed using a special seeding method that enabled singlecells to become confluent and passageable in 3–4 weeks. Atnon-permissive conditions (37° C. or 39° C., no γINF and low or no FBS)cell growth arrests.

Given the high transfection efficiencies already observed in the UCLs,these cells are grown and passaged in defined media that is serum free.Once this is established, these cells are lipofected with theIRES-GFP-POU4F3 encoding plasmid. Cultures are monitored for GFPexpression over 1–6 DIV. When periods of high GFP fluorescence areobserved in live cultures, the culture are fixed and prepared for POU4F3and calbindin ICC.

EXAMPLE 2 Overexpression of POU4F3 in Lesioned Organ of Corti Cultures.

Cultures from P7-P14 mice are established and lesioned with 1 mMneomycin for 2 DIV. The media are removed and the cultures lipofectedwith pIRES2-GFP-POU4F3 for 6 hr and recovered in fresh media for 1–6DIV. Cultures are aldehyde-fixed and processed for POU4F3 and calbindinimmunocytochemistry. pIRES2-GFP only lipofected cultures serve ascontrols. The presence of a triple labeled cell (positive for GFP,POU4F3, and calbindin immunoreactivity) indicates that POU4F3 is capableof promoting the adoption of a hair-cell phenotype in the lesioned organof Corti. Further determination of this phenotype is corroborated withantibodies against other hair-cell selective markers such as myosin 6and myosin 7a using polyclonal antibodies derived against theseproteins.

Expression of hair-cell specific markers such as myosin 6 and 7a areobserved in the embryonic mouse organ of Corti at E16, 2–3 days afterthe expression of the Brn 3.1 transcription factor begins at E13.5.

EXAMPLE 3 Regeneration of Inner Ear Hair Cells in p27^(Kip1) −/− Mice

Previous reports in p27^(Kip1) −/− and +/− mice showed qualitativeevidence of supernumerary hair cells (HCs) in both inner hair cell (IHC)and outer hair cell (OHC) regions (Chen, P., & Segil, N. Development126:1581–1590 (1999); Lowenheim, H., et al., Proc. Natl. Acad. Sci. USA.96:4084–4088 (1999)). Unfortunately, the significant dysplasia of thesurrounding supporting cells could very well account for some if not allof these observations. Therefore, the number of IHCs and OHCs in thecochlea of p27^(Kipl) −/−, +/− and +/+ mice was measured. To moreaccurately assess whether there was a true increase in HC number,several regions from the same cochlea were analyzed. Using a HC specificmarker, a myosin-VIIa antibody, a 20% increase in the number of IHCs inp27^(Kip1) −/− cochlea was observed when compared with that inp27^(Kip1) +/− and +/+ cochlea. However, there was no statisticallysignificant difference in the overall number of OHCs between p27^(Kip1)−/−, +/− and +/+ cochlea, except for a 10% increase in the number ofOHCs in one analyzed region (Table 1). Table 1 shows the number of haircells (n) in four-week old p27^(Kip1) +/+, +/−, −/− mice. Counts weremade from a 100 μm length of sensory epithelium from three differentlocations along the longitudinal axis of the organ of Corti. Distancecorresponds to degrees from the apical tip of the cochlea (±s.d.).Comparisons were made with the same hair cell region across +/+, +/− and−/−. Statistical significance was determined using ANOVA.

TABLE 1 Genotype Distance n IHC OHC +/+  90  6 13.0 ± 0.0 42.8 ± 2.5 +/− 90 16 13.1 ± 0.7 44.1 ± 2.9 −/−  90 11 16.0 ± 1.9* 43.5 ± 2.3 +/+ 180 6 13.3 ± 0.8 40.5 ± 5.2 +/− 180 16 13.2 ± 0.8 43.1 ± 2.2 −/− 180 1117.0 ± 1.3* 45.9 ± 3.3* +/+ 360  6 13.7 ± 0.8 41.7 ± 5.1 +/− 360 16 13.4± 0.5 43.1 ± 2.8 −/− 360 11 16.2 ± 1.8* 41.0 ± 4.6 *p-value < .001

To determine whether auditory hair cells were being produced after theonset of hearing at postnatal day 10, two-week old p27^(Kip1) −/−, +/−,+/+ mice (P10–12) received three daily systemic injections ofbromodeoxyuridine (BrdU; 30mg/kg/s.c), a nucleotide analog which isincorporated into proliferating cells during S phase. Mice were thenpermitted to recover for two-days or two-weeks without furtherinjections. BrdU positive HCs were identified with immunocytochemistryusing light and fluorescence microscopy. Cochlea were also labeled withantibodies against myosin-VI and myosin-VIIa. In two-week old p27^(Kip1)−/− cochlea that were recovered for 2d after the last injection, noBrdU/myosin-VIIa positive cells were observed among the BrdU positivecells. However, in four-week old p27^(Kip1) −/− cochlea that wererecovered for 14 days after the last injection, BrdU/myosin-VIIapositive HCs were observed. Most of those double labeled were IHCs. Thisqualitative finding is similar to our quantitative assessments of IHCsand OHCs numbers. p27^(Kip1) +/−, +/+ mice were completely devoid ofBrdU positive cells at 2d and 14d of recovery. These data are summarizedin Table 2. Table 2 shows the number (n) of BrdU labeled cells in theorgan of Corti of two- and four-week old p27^(Kip1) +/+, +/−, −/− miceinjected with BrdU or BrdU/Amikacin with 2d or 14d of recovery. Countswere performed on a 1000 μm length of sensory epithelium taken in theapical half of the cochlea (±s.d.). Proliferation in the BrdU groupswere compared across +/+, +/− and −/−. Proliferation in theBrdU/Amikacin group was compared with that in the BrdU only group of thesame genotype. Statistical significance was determined using ANOVA.

TABLE 2 Genotype n BrdU + 2d n BrdU/Amikacin + 2d +/+ 10  0.0 ± 0.0  0.0± 0.0 +/− 12  0.0 ± 0.0  4.7 ± 7.6 −/−  4 82.3 ± 11.0*  96.3 ± 9.5Genotype n BrdU + 14d n BrdU/Amikacin + 14d +/+ 10  0.0 ± 0.0 10  0.0 ±0.0 +/− 18  0.0 ± 0.0 10  10.5 ± 12.0* −/−  6 82.2 ± 22.5* 12 137.4 ±17.0* *p-value < .001

To ascertain whether auditory HCs could be regenerated, HCs werelesioned using systemic injections of amikacin sulfate (P7-P2) and theninjected with BrdU (P10–12). Mice were sacrificed either 2 d or 14 dafter the last injection. The effects of an amikacin lesioning were atleast two-fold. First, in both p27^(Kip1) −/− and +/− mice, the numberof BrdU positive cells increased following amikacin/BrdU vs BrdU alonetreatment. In p27^(Kip1) +/− cochlea, the numbers of BrdU labeled cellsincreased in the majority of specimens examined, however, not allp27^(Kip1) +/− cochlea displayed BrdU positive cells. Evidence of HCregeneration was confirmed by sectioning the labeled cochlea. Second, agreater number of BrdU positive HCs were observed following amikacinlesioning in p27^(Kip1) −/− cochlea. The majority of the BrdU positiveHCs appeared in the regions of the cochlea where the amikacin sulfatehad injured or killed HCs (in the basal half of the cochlea). No BrdUpositive cells were observed in wt cochlea following amikacin/BrdU orBrdU alone treatment. These data are summarized in Table 2.

To measure specific protein levels, single cochlear lysates wereserially diluted and run on polyacrylamide gels. Western blotting showedthat myosin-VI and VIIa levels appeared roughly equal across p27^(Kip1)−/−, +/− and +/+ cochlea, although a stronger myosin-VIIa band wasobserved from p27^(Kip1) −/− cochlea. p27^(Kip1) +/− cochlea containedapproximately 50% of the p27^(Kip1) protein levels found in wt cochlea,indicating that a reduction of p27^(Kip1) to 50% of normal can stimulatesupporting cell proliferation and allow some hair cell regeneration tooccur following amikacin sulfate treatment.

The protocol used to measure protein levels in the cochlea is asfollows. A cochlea is transferred to a tube with 10 μl of extractionbuffer which contains:HEPES (25 mM), NP-40 (0.7%), Aprotintin (1 mM),Leupeptin (1 μg/ml), Pepstatin (10 μM), phenylmethylsulfonylfluoride(PMSF) (1 mM), dithiothreitol (DTT) (1 mM), andethylenediaminetetraacetic acid (EDTA) (2 mM). The cochlea ishomogenized immediately and the tube is placed on ice for about 30 min.Add 5 μl of 4× sample buffer and adjust the salt concentration up to 0.5M. Heat up the sample to 90–100° C. for 5 min then spin at 13,000 rpmfor 10 min and collect the supernatant. Aliquots of protein from thesupernatant are run on a 15% SDS-PAGE gel for 50 min at 200 V and theproteins are transferred onto PVDF membrane for 1 hr at 100 V. Themembrane is blocked with 10% Amersham blocking buffer for 1 hr orovernight. Primary antibody in blocking buffer is applied to themembrane for 1 hr and the membrane is then washed five times withPBS/Tween for 5 minutes per wash. The membrane is probed with goatanti-mouse or goat anti-rabbit-alkaline phosphatase plus anti-biotin-APfor 1 hr and washed five times with PBS/Tween for 5 minutes per wash.

To determine whether p27^(Kip1) plays a similar role in the peripheralvestibular system, the proliferative capacity of the utricle, sacculeand cristae of p27^(Kip1) +/−, and +/+ mice was examined. Mice (P7-P12)received systemic injections of amikacin sulfate (500 mg/kg/d/s.c) forsix consecutive days that were combined with injections of thereplication marker, bromodeoxyuridine (BrdU; 30 mg/kg/d/s.c) betweenP10-P12. Mice were also injected with BrdU alone in a similar fashion.Mice were then sacrificed 14 d later and the vestibular sensory organswere fixed, dissected and processed for BrdU immunocytochemistry. BrdUpositive nuclei were counted from whole mounts using light microscopyand Nomarski optics. Selected organs were further processed for crosssection analysis.

In p27^(Kip1) −/− mice that received BrdU only, very low levels ofBrdU-labeled cells were observed in the saccule and utricle. However,after combined amikacin/BrdU treatment, a 40-fold increase in the numberof BrdU positive cells was observed in both organs. Approximatelyone-half of the labeled cells appeared as doublets suggesting recent orongoing cell divisions. Plastic cross sections showed that the majorityof the BrdU labeled cells were in the basal layer of the sensoryepithelium, along the basal membrane. BrdU positive HCs were alsoobserved in both otolithic organs. Most of these regenerated HC appearedas type I HCs in that they were contacted by a calyx. In p27^(Kip1) +/−mice that received BrdU only, no proliferation was observed in eitherthe saccule or utricle. After combined amikacin/BrdU treatment, a verylow level of proliferation was induced. In p27^(Kip1) +/+ mice, no BrdUpositive nuclei in either the saccule or utricle were observed afteramikacin/BrdU or BrdU alone. Interestingly, no BrdU positive cells wereobserved in the cristae of any genotype following amikacin/BrdU or BrdUonly. These data indicate significantly different effects in deletingp27^(Kip1) among the various vestibular sensory organs and between thevestibular sensory organs and the organ of Corti. These data aresummarized in Table 3. Table 3 shows the numbers of BrdU labeled cellsin the vestibular organs of four-week old p27^(Kip1) +/+, +/− and −/−mice injected with BrdU or BrdU/Amikacin and after a 14 d recovery.Counts were performed from whole utricular, saccular and cristae sensoryepithelium (±s.d.). In the BrdU group, statistical significance wasdetermined by comparing proliferation levels in the same sensory organacross +/+, +/− and −/−. In the BrdU/Amikacin group, statisticalsignificance was determined by comparing proliferation levels in thesame sensory organ of the same genotype. Statistical significance wasdetermined using ANOVA.

TABLE 3 genotype BrdU BrdU/Amikacin utricle (n)  8 +/+  0.0 ± 0.0  0.0 ±0.0 20 +/−  0.0 ± 0.0  0.4 ± 0.7 12 −/− 19.7 ± 13.4* 45.5 ± 19.2*saccule (n)  6 +/+  0.0 ± 0.0  0.0 ± 0.0 16 +/−  0.0 ± 0.0  1.6 ± 1.9  9−/− 32.7 ± 14.3* 51.8 ± 15.3* cristae (n) 12 +/+   0.0 ± 0.0  0.0 ± 0.012 +/−  0.0 ± 0.0  0.0 ± 0.0 12 −/−  0.0 ± 0.0  0.0 ± 0.0 *p-value <.001

Selected semi-thin sections were re-embedded in plastic, thin sectionedand examined under electron microscopy. BrdU positive HCs displayedstereociliary bundles, cuticular plates, calyceal innervation withevidence of synapse formation.

EXAMPLE 4 Antisense Inhibition of p27^(Kip1) Expression

To test whether inhibition of the p27^(Kip1) gene product in p27^(Kip1)+/+ cochlea would allow non-mitotic supporting cells to proliferate,wild type organ of Corti explants (P7-P10, at age of hearing onset) weretreated with p27^(Kip1) antisense oligonucleotides (ONs). Explantcultures were established by dissecting the organ of Corti from thecochlea, removing the tectorial membrane and adhering the organ of Cortito a glass slide coated with Cell-tak and maintained at 37° C. in a 5%CO₂ environment. The explant was then exposed to an ototoxic antibiotic(1 mM neomycin sulfate) for 48h which killed >95% of the HCs (Kil, J.,et al., ARO abs. 21:672 (1998)). The neomycin containing media was thenremoved and p27^(Kip1) antisense oligonucleotides (Ons) (40 nM) wereadministered using a cationic lipid (lipofection) for a period of 24–48hr. Some of these living cultures were examined under fluorescence todetect the presence of FITC conjugated antisense ON.

FITC-positive supporting cells were detected in 18–24 h and increased innumber and fluorescent intensity between 24–48 h. Cultures were aldehydefixed and processed for BrdU immunocytochemistry. BrdU-positivesupporting cells appeared in most cochlear cultures after 24 h ofantisense oligonucleotide (ON) treatment. BrdU-positive doubletsappeared in antisense ON treated cultures that were recovered for anadditional 24 hr without antisense ONs. This indicated that successfulcompletion of M phase and subsequent cell division could occur.Lipofection-only treated cultures contained very low levels ofBrdU-labeled supporting cells.

We observed that administration of p27^(Kip1) antisense oligonucleotidescan induce supporting cells to proliferate in wild-type cochlea. Thisobservation is unique and rather significant. Previous workdemonstrating p27^(Kip1) antisense ON induced proliferation were shownin actively dividing cells that had been transiently or reversiblygrowth arrested (Coats, S., et al, Science, 272:877–880 (1996); Dao, M.A., et al, Proc. Natl. Acad. Sci. USA, 95:13006–13011 (1998)). Ourresults are the first demonstration that terminally mitotic cells in aterminally differentiated organ can reenter the cell-cycle afterinhibition of p27^(Kip1) gene products.

Again, supporting cell proliferation normally ceases between E12–14 inthe mouse organ of Corti. After this point, supporting celldifferentiation continues through the second week of postnatal life. Atthe time of explant, cultures treated as described herein have alreadydeveloped some adultlike morphologic characteristics. These data furthersupport the role for p27^(Kip1) antisense ON as a potential means forinducing hair cell regeneration. In p27^(Kip1) +/− mice, a reduction to50% of normal protein levels allows some terminally differentiatedsupporting cells to overcome the p27^(Kip1) blockade and re-enter thecell cycle and proliferate.

The addition of growth factors that induce cell proliferation in otherepithelial organs, does not promote cell proliferation or hair cellregeneration in the postnatal organ of Corti, either in vitro or invivo. The experiments reported herein identify identified a ratherubiquitous and potent cell cycle inhibitor that, when deleted, allowsthe organ of Corti to regenerate some of its hair cells spontaneously.The organ of Corti of mice containing one copy of the gene and 50% ofnormal protein levels are capable of auditory hair cell regeneration.

In addition, organotypic cultures can be established on glass slides(Nunc) coated with CellTak (Collaborative Research) in 100 μls ofNeuroBasal media (Gibco) containing 1 mM neomycin. This treatment kills95% of the hair cells and also facilitates the level of transfection.Cultures are lipofected with antisense molecules using commerciallyavailable lipofection reagents (e.g, Perfect Lipofection Kit;InVitrogen, Inc.). The media also contains BrdU (10 μM) to identifyproliferating cells. In addition, various recombinant growth factorssuch as TGF-alpha (1–100 nM), insulin (10–100 μM) and IGF-1 (1–100 μM)can be used to increase or drive this proliferative effect.

EXAMPLE 5 The Use of p27^(Kip1) Antisense Oligonucleotides to StimulateCell Proliferation in a Guinea Pig Fibroblast Cell Line in Vitro

Cultures of cell lines were established that are responsive top27^(Kip1) antisense oligonucleotides (ON) during the period of serumwithdrawal and growth arrest. These cultures include a guinea pigfibroblast line (JH4). Lipofection of the 16 mer antisense ON (havingthe nucleic acid sequence set forth in SEQ ID NO:20) reversed growtharrest in the JH4 cell line to over 40% of normal.

EXAMPLE 6 Stimulation of Supporting Cell Proliferation in Guinea PigCochlea Using p27^(Kip1) Antisense Molecules

Two recent, independent studies indicate that antisense ONs can besuccessfully delivered through the perilymphatic space and elicitchanges in the organ of Corti of mature guinea pigs (D'Aldin, C., etal., Mol. Brain Res., 55:151–164 (1998); Leblanc, C. R., et al. Hear.Res., 135:105–112 (1999)). The presence of FITC-antisense ON againstspecific sequences to the mRNA of GluR2 was seen in the spiral ganglia,supporting cells, and inner and outer spiral sulcus cells within 24 hafter the osmotic pump installation. A subsequent selective decrease inGluR2/3 protein was also observed in situ (D'Aldin et al., 1998, supra).

The expression pattern of p27^(Kip1) in the mature guinea pig organ ofCorti was found to be similar to that observed in developing and adultmice. Supporting cell proliferation in guinea pig cochlea is stimulatedas follows:

General anesthesia is induced by inhalation of Isoflurane (5% for theinduction and 2–3% for the maintenance) or by intramuscular injection ofketamine (50 mg/kg) and xylazine (9 mg/kg). 1% lidocaine is injectedbehind the pinna locally. The animal is placed on a warming pad to keepbasal body temperature constant during the surgical operation.Respiration and circulation is monitored carefully. Drugs are deliveredinto the guinea pig's inner ear by surgically implanting an infusionunit which consists of a coiled catheter and an osmotic minipump (Alzet,cat no. 2002, 0.5 μl/h flow rate for up to 14 days). The coil is loadedwith the drug and the osmotic minipump carries a dye. The total volumeof drug pumped into the inner ear can be monitored by the amount of dyewhich is pumped into the coil. The drug is dissolved in a solution ofartificial perilymph which consists of: 137 mM NaCl, 5 mM KCl, 2 mMCaCl₂, 1 mM MgCl₂, 10 mM Hepes, 11 mM Glucose, pH 7.4 and osmolarity 300mOsm/L.

All surgical procedures are done under a dissection microscope and understerile conditions. The mastoid bulla (middle ear space) is exposed viaa post-auricular incision and opened using a 1 mm cutting burr to allowvisualization of the basal turn of the cochlea. A cochleostomy about 1mm inferior to the round window is fashioned using a 0.5 mm diamondpaste burr. Observed inner ear perilymph fluid leaking from this siteconfirms correct positioning of the cochleostomy. The tip of theinfusion unit is inserted into the cochleostomy and the tubing issecured on the wall of the middle ear by dental cement. The infusionunit is stored in a subcutaneous pocket created behind the neck. Theskin incision is closed in layers with 2–0 silk. This procedure isperformed on both ears to avoid any subsequent imbalance or rotationalbehavior and to reduce the number of animals needed to complete theexperiment. The procedure takes approximately 30 minutes per side.

Repeat surgery for changing the infusion unit is done under generalanesthesia one week after implantation. The procedure is done onlythrough the initial post-auricular skin incision and does not involvere-entry into the middle ear space. This procedure takes 5–10 minutesper infusion unit. The post-surgical condition of the animal ismonitored daily by checking activity, appetite, drinking, feces and bodyweight. The animal is sacrificed if it has lost more than 20% of itsbody weight post-surgery or exhibits severe rotational behavior or headtilt. No operative discomfort should occur and this procedure producesonly slight postoperative discomfort.

As shown in Table 4 below, normal controls are given a solution ofartificial perilymph for one or two weeks. The hair cell loss groupreceives gentamycin sulfate for one week to kill the hair cells withimmediate sacrifice and two week recovery. Three concentrations are usedthat should provide a total loss of HCs as well as a graded loss of HCs.Cationic liposomes in 5% (w/v) dextrose solution are delivered for oneto two weeks. The purpose of this group is to see if any damage iscaused by lipofection. Lipofection after hair cell loss involves thesubstitution of a gentamycin containing pump to a lipid containing pump.Lipofection plus antisense after hair cell loss involves lipofectingFITC-antisense for another week. Lipofection plus antisense plus growthfactors after damage are delivered for one week followed by thelipofection of antisense and growth factors for another week. Severalgroups involve combining antisense with growth factors, includingTGF-alpha, insulin, and IGF-1. In all animals, a separate osmotic pumploaded with BrdU is implanted subcutaneously to allow identification ofmitotically active cells

TABLE 4 Animals Drugs Concentration Delivery time of drugs (d) (n)(order) (range) Recovery time after (d) 12 GM/L 0.1–10 mg/ml 7 0, 7, 1412 GM/L/FITC- 10–20 nM 7 AS 7, 14 12 GM/L/AS 10–20 nM 7 7, 14 12GM/FITZ-AS 10–20 nM 7 or AS 7, 14 12 plus TGF-alpha 10–100 ng/ml 7 3, 7,14 12 plus Insulin 1–10 μg/ml 7 3, 7, 14 12 plus IGF-1 10–100 ng/ml 7 3,7, 14

Hair cell damage resulting from gentamycin is checked by myosin-VIIaimmunocytochemistry (a hair cell specific marker) and phalloidinhistochemistry (F-actin marker) using a whole mount staining technique.The transfection efficiency of liposome and p27^(Kip1) antisenseoligonucleotides is assessed by observing for the presence ofFITC-labeled nuclei under epifluorescence. BrdU immunohistochemistry isused to determine whether proliferation was induced with p27^(Kip1)antisense ON treatment. Selected specimens are analyzed under electronmicroscopy.

Some experiments are done using a double labeling with BrdU andmyosin-VIIa to assess the number of new supporting cells versus thenumber of total hair cells.

Other experiments are done using double labeling with BrdU and vimentinto assay the number of new supporting cells. Double labeling with BrdU,vimentin and myosin, distinguishes between the number of new hair cellscompared to the number of new supporting cells. The baseline for organof Corti supporting cell proliferation and auditory hair cellregeneration in mammals, is zero, making statistical significance easierto attain with a low number of observed events using one-way ANOVAs.

EXAMPLE 7 Assessment of Auditory Function After Ototoxic Insult and/orp27^(Kip1) Antisense Treatment in Guinea Pigs

Auditory Brainstem Responses (ABR) is tested in guinea pigs afterototoxic insult and/or p27^(Kip1) antisense treatment. ABR thresholdsare compared within the same animal over time and across animals fromthe same or different groups (pre- and post-surgical). Significance isdetermined using one-way analysis of variance (ANOVA) for each stimulusfrequency and intensity. Differences are considered statisticallysignificant for p-values <0.05. Previous studies have shown that thisoperation does not attenuate ABR responses post-operatively.

To record elicit ABRs, guinea pigs are anesthetized with Avertin (0.2ml/10 g body weight/i.m. using a 1.2% stock solution). The activeelectrodes are placed subcutaneously near the external meatus of the ear(0.1-mm silver wire; Narishige). The dural reference electrode is placedin a drilled hole rostral to the bregma (or an insert earphone into theear canal and the sound delivery tube secured to the pinna with surgicaltape). The ground electrode (Ag/AgCl₂ pellet) is fixed on the back. Thesound stimuli is either a broad band click of 100 μs duration or a 10 mstone burst (1 ms rise/fall time). Guinea pigs are in a sound attenuatedchamber (TDT model AC-1). The responses are measured and recorded via anAuditory Evoked Response Workstation (SmartEP; Intelligent HearingSystem). Guinea pigs are presented with a stimulus intensity seriesincremented from 20 to 85 dB in 5 dB steps for both click and tone burststimuli. For tone bursts, a stimulus frequency series (1, 2, 4, 8, 16,32 Hz) at a constant intensity of 50 dB is also used. Stimuli arerepeated 5 times/s and a total of 512 trials will be averaged. Thresholdis defined as the lowest intensity capable of eliciting a replicable andvisually detectable ABR.

EXAMPLE 8 Improvement in Auditory Function in Amikacin Sulfate-Treatedp27 Heterozygote Mice

Experimental animals were treated the same as the mice described in theexperiments reported in Table 2 herein, except that, in the presentexample, there was an additional recovery time point at four weeks afteramikacin treatment. Auditory function was measured using the auditorybrainstem response (ABR) using subcutaneous recording electrodes placedon three head points in Isofluorane anesthetized mice. The soundintensity threshold was determined by presenting single frequencies asdifferent sound intensities (intensity measured in decibels). The higherthe tone intensity that is required to elicit the ABR, the higher theauditory threshold, i.e., the worse the auditory function. The data setforth in FIGS. 3–6 shows auditory improvement in five out of eight p27heterozygotes (p<0.001).

In Table 2 herein, five out of ten p27 heterozygotes showed evidence ofinner cell proliferative regeneration as assessed by BrdU-labeling andmorphologic criteria. These data showed that the majority ofBrdU-labeled cells were supporting cells, not hair cells. The improvedauditory function in the p27 heterozygotes may, therefore, be due toregeneration of supporting cells either alone, or in combination withregeneration of hair cells.

EXAMPLE 9 The Lipofection Method of Gene Delivery and Gene Expression inthe Mouse Organ of Corti Culture System

Lipofecting the organ of Corti utilized cochlear explants obtained frompostnatal day 7–14 mice grown for a total of up to 8 days in vitro(DIV). Cultures were grown in defined culture media composed ofNeurobasal Media with B27 supplement (Gibco). Cultures were exposed toan aminoglycoside antibiotic (1 mM neomycin sulfate for 48 hrs) whichselectively killed the inner ear sensory hair-cells. Eight differentlipid combinations were then tested from the Perfect Lipofection Kit(InVitrogen). A bacterial plasmid encoding a betagalactosidase reportergene driven by a CMV immediate/early gene promoter (InVitrogen) wasdelivered over a 6 hr period. The cultures were aldehyde-fixed andprocessed for betagalactosidase expression using x-gal histochemistry.X-gal labeling appeared in supporting-cells (54.3+/−15.3 labeled cellsper 1000 μm length in the regions of the sensory epithelium that oncecontained hair-cells, versus 5–10 labeled cells per 1000 μm length intissue that had not been lesioned).

Given the labor involved in detecting beta-galactosidase expression, thesize of the betagalactosidase encoding construct (i.e., 4.1 kbp) and thereduced compatibility of this technique with other desired ICCprocedures, cultures are lipofected with a plasmid encoding GreenFluorescent Protein (GFP; Clonetech). Detection of GFP requires astandard FITC filter set (excitation maximum 488 nm, emission maximum509 nm) and has been successfully transfected into cochlear hair-cells,supporting-cells and neurons using an AAV vector system.

Organ of Corti cultures established from P7–P14 Swiss Webster micederived from our breeding colony are lipofected using a variety ofcommercially available lipofection reagents (i.e., FuGENE TransfectionReagent; Boehringer-Mannheim). These efficiences are compared againstthe transfection efficiences achieved by the InVitrogen Kit. Thesuperior lipofection reagent and the superior lipid to DNA ratio (3:1,6:1, 9:1) are determined by counting the number of GFP-positive cellswithin the organ of Corti along a 1000 μm length taken at the middle ofthe explant. Cells are visualized using a Nikon epifluorescencemicroscope equipped with a CCD digital camera that outputs imagesdirectly into an imaging software program where cell counts areperformed.

An aminoglycoside antibiotic lesion of the hair-cells is then becombined with subsequent lipofection. Media containing 1 mM neomycinsulfate (Sigma) is administered to kill the hair-cells over a 48 periodin culture. Unlike cultures derived from neonatal mice, the two-week oldmouse which has developed auditory function is more easily affected bythis concentration of neomycin resulting in a greater than 95% loss ofhair-cells as determined by calbindin immunoreactivities and plasticcross-section analysis. The remaining supporting-cells are lipofectedfor 6 hr with a GFP encoding plasmid. Cultures are rinsed and grown infresh media for an additional 1–4 DIV for a total of 3–6 DIV. Culturesare aldehyde-fixed and GFP is visualized directly under epifluorescence.Several reports have demonstrated a loss of GFP fluorescence afteraldehyde fixation. This may necessitate the use of a commerciallyavailable GFP antibody (Clonetech) to enhance the fluorescence oflipofected cells.

EXAMPLE 10 Excision and In Vitro Culture of Mouse Inner Ear

The inner ear of a mouse was excised in the following manner. Postnatalday 7–14 Swiss Webster mice were decapitated and their skulls immersedin 70% ethanol for 5 min to disinfect. Under sterile conditions, theskull was cut into halves along the mid-sagittal axis and placed into 3ml of culture media (Neuralbasal™ Media at pH 7.4; Gibco) in a 35 mmplastic culture dish (Nalge Nunc International, 2000 North Aurora Road,Naperville, Ill. 60563). Using surgical forceps, the bony inner earlabyrinth was visualized and separated from the temporal bone. Theoverlying connective tissue, stapes bone, facial nerve and stapedialartery were removed. Using a fine forcep, a small hole about 2 mm indiameter was made through the apical turn of the lateral cochlear wall.This surgically created conduit, along with the patent oval and roundwindows of the cochlea, permit ready diffusion of the culture media intothe fluid-filled inner ear.

Typically, an inner ear excised and prepared in the foregoing manner istransferred to the HARV™ or CCCV™ vessel which contains 50 or 55 ml ofNeuralbasal™ Media supplemented with either N2 or B27 media supplement(both sold by Gibco-BRL, Catalogue number 17504–036), 10 U/ml ofpenicillin and 0.25 μg/μl of fungizone. The B27 supplement is sold as a50× concentrate which is used at a working concentration of 0.5× (e.g.,550 μl of 50×B27 stock solution is added to 55 ml of Neuralbasal™Media). The N2 supplement stock solution is 100× and is used at aworking concentration of 1×(e.g., 550 μl of 100×N2 stock solution isadded to 55 ml of Neuralbasal™ Media). The vessel is then placed in atissue culture incubator at 37° C. and in a 95% air/5% CO₂ environment.The vessel is then rotated at 39 rpm for periods of 24–168 hr. 50% mediachanges are made every 48 hr. As few as 2 and as many as 12 inner earshave been successfully cultured in one vessel.

To lesion the inner ear sensory hair-cells, the inner ear is placed inNeuralbasal™/N2 or B27 media that contain 1 mM neomycin sulfate (Sigma,P.O. Box 14508, St. Louis, Mo. 63178) for 24–48 hr. After this cultureperiod, the media is completely replaced with media devoid of neomycin.

EXAMPLE 11 Culture Media

Table 5 shows the composition of Neuralbasal™ medium (1×) sold by Gibco.All concentrations are working concentrations, i.e., the concentrationsof the components in the medium in which the fluid-filled sensory organis incubated.

TABLE 5 Neuralbasal ™ media composition Component mg/liter μM Inorganicsalts CaCl₂ (anhydrous) 200 1,800 Fe (NO₃)₃ 9H₂O 0.1 0.2 KCl 400 5,360MgCl₂ (anhydrous) 77.3 812 NaCl 3,000 51,300 NaHCO₃ 2,200 26,000NaH₂PO₄H₂O 125 900 D-glucose 4,500 25,000 Phenol Red 8.1 23 HEPES 2,60010,000 Sodium Pyruvate 25 230 Amino Acids L-alanine 2.0 20 L-arginineHCl 84 400 L-asparagine H₂O 0.83 5 L-cysteine 1.21 10 L-glutamateGlycine 30 400 L-histidine HCl H₂O 42 200 L-isoleucine 105 800 L-lysineHCl 146 5 L-methionine 30 200 L-phenylalanine 66 400 L-proline 7.76 67L-serine 42 400 L-threonine 95 800 L-trptophan 16 80 L-tyrosine 72 400L-valine 94 800 D-Ca pantothenate 4 8 Choline chloride 4 28 Folic acid 48 i-Inositol 7.2 40 Niacinamide 4 30 Pyridoxal HCl 4 20 Riboflavin 0.410 Thiamine HCl 4 10 Vitamin B12 0.34 0.2

The following antibiotics may be added to Neuralbasal™ medium. Fungizonereagent (amphotericin B, 0.25 μg/ml, and sodium desoxycholate, 0.25μg/ml) which is sold by Gibco-BRL, Catalog number 17504-036. PenicillinG (10 units/ml) which is sold by Sigma, Catalog number P 3414. Neomycinsulfate (1 mM), sold by Sigma, Catalog number N 6386. Neuralbasal™medium may also be supplemented with L-Glutamine (2 mM).

EXAMPLE 12 Assay For Sensory Epithelium Vitality During Long TermCulture

In the practice of one aspect of the present invention, themicrogravitational environment provided by the rotation of a culturevessel allows the sensory epithelium of the inner ear to be maintainedfor prolonged periods of culture (>168 hr.) without significantdegradation or loss of the sensory hair-cells or non-sensorysupporting-cells. Data demonstrating the continued vitality of thesensory hair cells during prolonged culture were obtained by labelingthe sensory epithelia with a probe against F-actin (phalloidin-FITC)that labels the surfaces of sensory and non-sensory cells, and with ahair-cell specific antibody against calbindin, a calcium bindingprotein. Both labels were detected and photographed underepifluorescence microscopy.

Cross-sectional data indicated that the normal cytoarchitecture of theinner ear sensory epithelia are maintained. For example, the Organ ofCorti has several fluid-filled spaces called the tunnel of Corti andspaces of Nuel that are necessary for normal auditory function. Thesespaces occur between hair-cells and supporting-cells and are maintainedafter prolonged periods of culture. In normal gravitationalenvironments, (i.e., when the inner ear is floated without rotating theculture vessel) the sensory epithelia begin to degenerate. Withoutrotation, within 24 hr the hair-cells are either completely missing orappear to be undergoing various endstages of cell death. After 48 hr.,the supporting-cells are completely missing, or are present but with thetotal loss of the tunnel of Corti and spaces of Nuel. Rotating thevessel prevents this degradation and maintains normal cytoarchitecture.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method for stimulating the formation of an inner ear sensory haircell from an inner ear support cell in vivo, the method comprising thesteps of: (a) damaging a first inner ear sensory hair cell by contactingthe sensory hair cell, in vivo, with an effective amount of anantibiotic; and (b) locally introducing into an inner ear support cell,that is in contact with the damaged sensory hair cell, a nucleic acidmolecule that is complementary to a portion of an mRNA molecule thatencodes p27Kip1 and inhibits mammalian p27Kip1.
 2. The method of claim1, wherein the support cell is selected from the group consisting ofHensen's cells, Deiter's cells, inner Pillar cells, border cells andouter Pillar cells.
 3. The method of claim 1, wherein the antibiotic isan aminoglycoside.
 4. The method of claim 1, wherein the antibiotic isutilized at a concentration of from about 100 mg/kg/d to about 1000mg/kg/d.
 5. The method of claim 1, wherein the antibiotic is deliveredto the inner ear by injection.
 6. The method of claim 1, wherein theantibiotic is delivered to the inner ear through a cannula.
 7. Themethod of claim 1, wherein the nucleic acid molecule hybridizes understringent conditions to a nucleic acid molecule encoding mammalianp27Kip1.
 8. The method of claim 1, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule comprising SEQ ID NO:8, under ahybridization stringency greater than 2×SSC at 55° C.